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Biomedical subjects

R Teshima

Publications and source records attributed to R Teshima.

At least 73 records · Page 4Linked to original sources
PubMed1998

Inhibitory effects of apple polyphenol on induced histamine release from RBL-2H3 cells and rat mast cells.

The anti-allergic activities of polyphenol fractions extracted from immature fruits of apple (Rosaceae, Malus sp.) were evaluated by in vitro assays. A crude apple polyphenol (CAP) fraction, which had been obtained from the juice of immature apples by reverse-phase column chromatography, was further purified by LH-20 column chromatography to obtain an apple condensed tannin (ACT) fraction consisting of linear oligomeric epicatechins from the dimer to pentadecamer. ACT strongly inhibited the release of histamine from rat basophilic leukemia (RBL-2H3) cells stimulated by the antigen-stimulation and from rat peritoneal mast cells stimulated by compound 48/80. The IC50 values for histamine release were 30 micrograms/ml and 25 micrograms/ml, respectively. ACT also inhibited hyaluronidase activity and the increase in intracellular free calcium concentration in RBL-2H3 cells stimulated with the antigen. These results suggest that ACT affected early signal transduction including the calcium influx.

Animals↗
PubMed1997

Effect of an ectokinase inhibitor, K252b, on degranulation and Ca2+ signals of RBL-2H3 cells and human basophils.

We examined the effects of K252b, an ectoprotein kinase inhibitor of microbial origin, on the activation process of RBL-2H3 cells by cross-linking of IgE receptors by the endoplasmic reticulum Ca2+-ATPase inhibitor 2,5-di(tert-butyl)-1,4-hydroquinone or by the Ca2+ ionophore A23187. Analysis of phosphorylation of ectoproteins following IgE receptor cross-linking revealed that K252b mainly inhibited the phosphorylation of a 130-kDa protein. The inhibitor simultaneously inhibited degranulation and the sustained increase in the cytosolic calcium ion concentration even after addition of Ag. In contrast, K252b did not inhibit the increase in degranulation and cytosolic calcium ion concentration caused by stimulation with 2,5-di(tert-butyl)-1,4-hydroquinone and A23187. Permeation of K252b into RBL-2H3 cells, assessed by fluorescence intensity, was very low. K252b also inhibited degranulation caused by IgE receptor cross-linking in human basophils, but did not inhibit the degranulation caused by A23187. Thus, our findings suggest that the effects of K252b may be mediated by outer surface-bound or -anchored K252b-sensitive molecules on RBL-2H3 cells and human basophils, and that the phosphorylation of ectoprotein may involve a transmembrane influx of Ca2+ by IgE receptor cross-linking.

Basophils↗
PubMed1997

Analysis of heat shock proteins and cytokines expressed during early stages of osteoarthritis in a mouse model.

OBJECTIVE: Osteoarthritis (OA) is a debilitating disease of the joints. The joints of affected individuals are characterized by a progressive degeneration of articular cartilage leading to inflammation and pain. The expression of heat shock proteins (HSPs) is a ubiquitous self-protective mechanism of all cells under stress, furthermore, the synovium of osteoarthritic individuals contains high levels of cytokines. This study seeks to establish the role of HSPs and cytokines in OA. METHODS: We have investigated the presence of HSPs and cytokines in articular cartilage during early stages of OA in a mouse that is known to develop spontaneous OA lesions (C57 black mouse). The articular cartilage from closely related mice (C57BL/6) was used as control. Messenger RNAs (mRNAs) for HSPs (HSP32, HSP47, HSP60, HSP70, HSP84 and HSP86) and cytokines [interleukin-1 beta (IL-1 beta), interleukin-6 (IL-6), tumor necrosis factor-alpha (TNF-alpha) and interferon-gamma (IFN-gamma)] were detected by reverse transcription-polymerase chain reaction (RT-PCR). RESULTS: The mRNA levels of HSP47, HSP70, HSP86, IL-6, and IFN-gamma were up-regulated in the cartilage of C57 black mice, whereas, the level of expression of HSP32, HSP60, HSP84 and IL-1 beta remained unchanged. Furthermore, the expression of IL-1 beta, IL-6, TNF-alpha and IFN-gamma mRNA was associated with expression of HSP60, HSP47, HSP70 and HSP70/HSP86 mRNA, respectively. CONCLUSIONS: The findings in this study suggest that chondrocytes are conditioned under non-physiological stress during early stages of OA, In addition, among HSPs, HSP70 was associated with two different highly expressed cytokines in C57 black mice, indicating the possible role of HSP70 as a characteristic indicator of early stage of OA.

Animals↗
PubMed1996

Confocal fluorescence microscopy for antibodies against a highly conserved sequence in SH2 domains.

We have prepared monoclonal antibodies for a highly conserved sequence (GTFLVRESETTK) in SH2 domains. Mouse IgG1s (12E and 32D) prepared against a peptide-conjugated keyhole lympet hemocyanin specifically bound the antigenic peptide but not the carrier protein. Western blot analysis showed that one IgG1 (12E) recognized mainly 62 kDa proteins (possibly src-family tyrosine kinases) from triton X-100 extracts of RBL-2H3 cells and that another (32D) recognized mainly 32 and 110 kDa proteins. Confocal fluorescence microscopy showed that the SH2 domains had a diffuse cytoplasmic distribution and were not present in the nucleus. Following antigen stimulation, a markedly different cellular distribution was observed in the cells stained with 12E and 32D IgGs. 12E IgGs strongly stained the plasma membranes while 32D IgGs stained small granules in the cytoplasm. As 12E IgGs bound 62 kDa proteins on Western blotting, the results suggest that tyrosine kinases cluster along the plasma membranes and/or than conformational changes occur in the domains after antigen stimulation.

Amino Acid Sequence↗
PubMed1996

Effects of hydroquinone-type and phenolic antioxidants on calcium signals and degranulation of RBL-2H3 cells.

We previously reported that a hydroquinone-type antioxidant, 2,5-di(tert-butyl)-1,4-hydroquinone (DTBHQ), increases intracellular free Ca2+ concentration ([Ca2+]i), causes degranulation together with a protein kinase C activator, phorbol 12-myristate 13-acetate (TPA), and increases antigen-induced degranulation in rat basophilic leukemia (RBL-2H3) cells. In this study, the effects of five-hydroquinone-type and phenolic antioxidants (2,5-di(tert-amyl)-1,4-hydroquinone [DTAHQ], 2-tert-butyl-1,4-hydroquinone [MTBHQ], 3,5-di(tert-butyl)-4-hydroxytoluene [BHT], 3,5-di(tert-butyl)-4-hydroxyanisole [DTBHA], and 3-tert-butyl-4-hydroxyanisole [MTBHA]) on [ca2+]i and degranulation (beta-hexosaminidase release) were examined and compared with that of DTBHQ. DTAHQ (> or = 3 microM) showed effects similar to those of DTBHQ (10 microM) on [Ca2+]i elevation, induction of degranulation with TPA, and increase of antigen-induced degranulation. BHT (50 microM) and DTBHA (50 microM) caused [Ca2+]i elevation and increased degranulation in the presence of TPA or antigen, but their effects were less than those of DTBHQ and DTAHQ. MTBHQ and MTBHA had no effect on [Ca2+]i and degranulation, even at 50 microM. The degree of Ca2+ response caused by the compounds correlated well with the increase in degranulation, but not with their antioxidant activity estimated with the first oxidation potential. From these results, it is suggested that the increasing effects of six antioxidants on degranulation in the presence of TPA or antigen were dependent on [Ca2+]i increase caused by the compounds, probably through their ability to inhibit endoplasmic reticulum Ca2+-ATPase.

Animals↗
PubMed1996

Surface expression of CD63 antigen (AD1 antigen) in P815 mastocytoma cells by transfected IgE receptors.

The surface expression CD63 antigen in rat basophilic leukemia cells (RBL-2H3) was observed after antigen stimulation by confocal fluorescence microscopy. The surface expression of CD63 antigen reflected the degranulation in RBL-2H3 cells. Then, we did the same experiments in P815 mastocytoma cells with transfected IgE receptors. The expression was observed in P815 cells with normal IgE receptors, but not in P815 variant cells with IgE receptors which were missing a C-terminal cytoplasmic domain of beta or gamma subunit. In addition, the expression in P815 cells with normal IgE receptors was mostly blocked by the pretreatment of herbimycin A. The results suggested that tyrosine phosphorylation of the C-terminal cytoplasmic domains of beta and gamma subunits was essential for degranulation.

Animals↗
PubMed1996

Effects of three different Ca(2+)-ATPase inhibitors on Ca2+ response and leukotriene release in RBL-2H3 cells.

The effects of three Ca(2+)-ATPase inhibitors, thapsigargin (TG), cyclopiazonic acid (CPA), and 2,5-di(tert-butyl)-1,4-hydroquinone (DTBHQ), on the Ca2+ response, degranulation, and leukotriene C4 (LTC4) release in RBL-2H3 cells were investigated. All three compounds elevated the intracellular free Ca2+ concentration ([Ca2+]i), and caused degranulation in the presence of 12-O-tetradecanoylphorbol-13-acetate (TPA), a protein kinase C activator. The dose-dependency of each compound in the Ca2+ response was in good agreement with that in degranulation. TG and CPA also caused the release of LTC4 in a dose-dependent manner, and this effect was unaffected by TPA or calphostin C, a selective PKC inhibitor. DTBHQ, however, did not induce LTC4 release, and rather inhibited the antigen-induced release of LTC4. These results suggest [1] that both degranulation and LTC4 release caused by these compounds are dependent on their [Ca2+]i increasing effect, [2] that degranulation and LTC4 release are mediated via independent pathways following the Ca2+ response, and [3] that DTBHQ additionally prevents the synthesis of LTC4 possibly by inhibition of 5-lipoxygenase.

Animals↗
PubMed1996

The effect of okadaic acid on histamine release, cell morphology and phosphorylation in rat basophilic leukemia (RBL-2H3) cells, human basophils and rat peritoneal mast cells.

To study the involvement of serine/threonine phosphatase in the signal transduction of mast cells, we examined the effects of okadaic acid (OA), an inhibitor of type-1 and -2A phosphatase on histamine release, cell morphology, calcium influx and protein phosphorylation of rat basophilic leukemia (RBL-2H3) cells, human basophils and rat peritoneal mast cells. OA inhibited IgE-mediated histamine release from RBL-2H3 cells and human basophils dose-dependently. There was a remarkable enhancement of IgE-mediated histamine release when rat peritoneal mast cells were suboptimally challenged. OA induced a marked change of cell features, detached RBL-2H3 cells from plastic well and kept the 18- and 68-kD proteins phosphorylated. These findings show that phosphatase may play a role in the modulation of secretion in mast cells.

Animals↗
PubMed1996

Quantitative evaluation of passive cutaneous anaphylaxis (PCA) using a hand-held spectrophotometer.

A hand-held spectrophotometer was used for the quantitative evaluation of PCA caused by anti-TNP-IgE and TNP-BSA. A good relationship existed between the dilution of sera and the value of delta E*ab (a numerical expression of color) obtained by a hand-held spectrophotometer. In addition, the value of delta E*ab and the amount of Evans blue measured by the conventional colorimetric method correlated well. Because the method using a hand-held spectrophotometer provides a simple and objective analysis, it appears that the method is suitable as a substitute for the conventional method, which is time-consuming and requires killing animals cruelly.

Animals↗
PubMed1995

Effects of 2,5-di(tert-butyl)-1,4-hydroquinone on intracellular free Ca2+ levels and histamine secretion in RBL-2H3 cells.

The effects of 2,5-di(tert-butyl)-1,4-hydroquinone (DTBHQ) on the intracellular free Ca2+ level ([Ca2+]i) and histamine secretion of rat basophilic leukemia (RBL-2H3) cells were examined. DTBHQ (0.1-10 mumol/l) alone induced rapid and sustained increases in [Ca2+]i in a concentration-dependent manner. In cells sensitized with anti-dinitrophenyl IgE, DTBHQ (10 mumol/l) further increased the antigen (dinitrophenylated BSA)-induced Ca2+ response. In the absence of external Ca2+ with addition of 1 mmol/l EGTA, both DTBHQ (10 mumol/l) and the antigen (10 microgram/ml) induced transient increase in [Ca2+]i. In sensitized cells, both DTBHQ (10 mumol/l) and antigen (10 micrograms/ml) elicited histamine secretion, although the response was far stronger in the latter case. The DTBHQ-induced histamine secretion was markedly enhanced by addition of the protein kinase C activator, phorbol 12-myristate 13-acetate (TPA) (10 ng/ml) whereas TPA alone did not cause any increase. Moreover, DTBHQ enhanced the antigen-induced histamine secretion. The results suggest that DTBHQ increases [Ca2+]i and enhances antigen-induced histamine secretion while DTBHQ alone does not cause as much histamine secretion as antigen, which support the idea that calcium signals are necessary but are not sufficient for maximum histamine secretion in RBL-2H3 cells.

Adenosine Triphosphatases↗
PubMed1995

Knee deformities in multiple hereditary exostoses. A longitudinal radiographic study.

For a mean period of 7.8 years, the morphology of knee deformity and its annual changes in 8 patients (16 knees; mean age at the initial examination, 8.4 years) with multiple hereditary exostoses were evaluated radiographically. Knee deformity developed during the growth spurt period. Genu valgum with a femorotibial angle that decreased more than 2 standard deviations from the mean of age-matched normal children was observed in 6 of the 16 knees. In 3 of these 6 knees, recurrent dislocation of the patella was observed. Angulation of the femur varied among the knees, but the tibia showed valgus angulation in the proximal metaphysis in all knees. Thus, knee deformity in patients with multiple hereditary exostoses was primarily genu valgum caused by valgus angulation of the tibia.

Child↗
PubMed1995

Structure of the most superficial layer of articular cartilage.

We studied the most superficial layer of macroscopically normal articular cartilage obtained from human femoral heads, using polarising microscopy and SEM. The most superficial layer, 4 to 8 microns thick, was acellular consisting of collagen fibrils. This layer could be peeled away as a thin film, with no broken collagen fibrils on its inferior surface or on the surface of subjacent cartilage layers. The orientation and diameter of collagen fibrils were different on these two surfaces. Our findings suggest that the most superficial layer is an independent one which is only loosely connected to the fibrous structure in the layer deep to it.

Cartilage, Articular↗
PubMed1994

Thapsigargin-induced nuclear calcium signals in rat basophilic leukaemia cells.

By a confocal fluorescence microscope with an argon-ion laser (488 nm) and a He-Cd laser (325 nm) we have studied thapsigargin-induced calcium signals in individual rat basophilic leukaemia (RBL-2H3) cells. In the presence or absence of external calcium ions, thapsigargin-induced calcium signals were transferred to the nucleus as well as to the cytoplasm of RBL-2H3 cells. The calcium signals were generally much stronger in the nucleus than in the cytoplasm. However, some of the RBL-2H3 cells had apparently reduced nuclear calcium signals. They had a basophil-like bilobed (multilobed) nucleus, although most RBL-2H3 cells had a mast-cell-like monolobed nucleus. In the cells with a bilobed nucleus, IgE-receptor-mediated calcium signals were neither transferred to the nucleus nor to the cytoplasm. The results gave a new insight into the understanding of the mechanism of the nuclear calcium signals in RBL-2H3 cells.

Animals↗
PubMed1994

Stimulatory effect of pervanadate on calcium signals and histamine secretion of RBL-2H3 cells.

We examined the effect of pervanadate on the activation of rat basophilic leukaemia (RBL-2H3) cells. The pervanadate, generated from a combination of H2O2 and vanadate (Vi), induced concomitantly protein tyrosine phosphorylation, formation of inositol 1,4,5-trisphosphate (IP3), an increase in [Ca2+]i, and histamine secretion in RBL-2H3 cells. These effects were clearly dependent on the ratio of H2O2/Vi. The secretion of histamine, IP3 formation, and sustained increase in [Ca2+]i were effectively induced by treatment of the cells with the pervanadate produced from 1 mM H2O2 and 1 mM Vi. These effects mimic the stimulatory effects of an antigen (dinitrophenylated BSA) on Ca2+ signals, histamine secretion and morphological changes. Protein tyrosine phosphorylation, formation of IP3 and transient increase in [Ca2+]i were markedly induced by the pervanadate produced from 3 mM H2O2 and 1 mM Vi. However, histamine secretion induced by the pervanadate was very low. After the pervanadate from 3 mM H2O2 and 1 mM Vi was treated with catalase, it was able to induce the [Ca2+]i increase and histamine secretion as much as the antigen did. This indicates that pervanadate from a lower H2O2 concentration (1 mM H2O2/1 mM Vi) and catalase-treated pervanadate from a higher H2O2 concentration (3 mM H2O2/1 mM Vi) are able to mimic the activity that was caused by cross-linking of IgE receptors with antigen. The present results also demonstrate that protein tyrosine phosphorylation seems to have a crucial role in Ca2+ entry from the external medium, and that a sustained [Ca2+]i increase is an important step for histamine secretion in RBL-2H3 cells.

Animals↗
PubMed1994

Effects of herbimycin A and ST638 on Fc epsilon receptor-mediated histamine release and Ca2+ signals in rat basophilic leukemia (RBL-2H3) cells.

We examined the effect of the two protein tyrosine kinase inhibitors, alpha-cyano-3-ethoxy-4-hydroxy-5-phenylthiomethylcinnamide (ST638) and herbimycin A, on the activation processes of rat basophilic leukemia (RBL-2H3) cells by cross-linking of IgE receptors. RBL-2H3 cells sensitized with DNP-specific monoclonal IgE antibody were stimulated with multivalent antigen (DNP conjugate of bovine serum albumin). Analysis of phosphotyrosine-containing proteins in their lysates by SDS-PAGE and immunoblotting revealed that these two inhibitors efficiently inhibited the tyrosine phosphorylation of several proteins (32, 42, 56, 66, 72, 92, 150 kDa) including phospholipase C-gamma 1. The inhibitors also caused parallel inhibitions of the histamine release, the formation of inositol 1,4,5-trisphosphate, and the increase in cytosolic calcium ion concentration at the late sustained phase. A digital imaging fluorescence microscopic analysis of antigen-dependent calcium signals in individual cells showed that these two tyrosine kinase inhibitors inhibited the calcium influx from the external medium more powerfully than the mobilization of calcium ion from internal stores. In contrast, the inhibitors did not affect the increase in the cytosolic calcium ion concentration or the histamine release induced by the calcium ionophore A23187. Taken together, our results suggest that tyrosine phosphorylation following antigen stimulation regulates phosphatidylinositol hydrolysis and the influx of extracellular calcium.

Animals↗
PubMed1994

Ligand-induced internalization and phosphorylation-dependent degradation of growth hormone receptor in human IM-9 cells.

The human growth hormone (hGH) induced a marked reduction in the number of human growth hormone receptors (hGHR) within 60 min, as assessed by immunoblotting of the crude membrane fraction from human IM-9 cells, without an increase in soluble forms of hGHR. The disappearance of hGH-induced hGHR was markedly inhibited by reagents that raise the internal pH of acidic organella and partially by protease inhibitors. These results suggest that hGH stimulation results in degradation of internalized hGHRs, where proteases in acidic compartments such as lysosomes may be involved. The relationship between the hGH concentration and the number of residual cell surface hGHRs 60 min after hGH stimulation yielded a curve with an inverted bell shape showing maximum internalization at 10 nM hGH. A similar relationship was shown in the hGHR degradation. The fact that the ligands in excess gave reduced internalization and degradation supports the idea that dimerization of hGHRs on the cell surface through the bivalent ligand hGH is required for their internalization and subsequent degradation. Following hGH stimulation, several hGHR-associated proteins including JAK2 were phosphorylated. These phosphorylations were inhibited by pretreatment with a protein kinase inhibitor, staurosporine. The hGHR internalization, however, was not markedly affected by the inhibitor. In contrast, the staurosporine inhibited the degradation of hGHR in a dose-dependent manner. These results suggest that staurosporine-sensitive phosphorylation is not required for the hGHR internalization, but the phosphorylation is involved in the degradation of hGHR.

Alkaloids↗